The present invention relates to a biotinylating reagent used in a purification procedure for synthesized peptide or proteins. The present invention will be a useful technique for making a pharmaceutical or a physiologically active agent.
Peptides and proteins are biological molecules existing normally in organisms. The elucidation of physiological activities and of mechanisms of these biological molecules are of much interest in the fields of biochemistry, physiology and medicine.
The synthesis of peptides or proteins having specific amino acid sequences has recently been made convenient the use of automated peptide synthesizers. Research in the above mentioned fields is expected to show much progress, if the synthesized peptides or proteins having specific amino acid sequences are provided with higher purities. However, the known peptide synthesis methods produce relatively large amounts of impurities in addition to the target compound. Therefore the most pressing objective of the solid-phase peptide synthesis method is to efficiently separate the target peptide from impurities in high yield (Analytical Biochemistry Vol. 170, p501, (1988)).
Gel filtration, high-performance liquid chromatography, or the combination thereof have been used for the purification of peptides or proteins synthesized by a solid-phase method (R. B. Merrifield, J. Am. Chem. Soc., 85, 2149 (1963)). Regarding some kinds of special peptides, affinity chromatography may be an effective purification method, but not a perfect one. The reason is that various amino acid deleted peptides may have an affinity (even a low degree) for the supports used in chromatography, the amino acid deleted peptides being synthesized as Impurities during the solid-phase synthesis and being in the resultant peptide mixture.
The solid-phase synthesis of peptides is made by a stepwise elongation. For example, in the case of the preparation of a 50 residue peptide wherein each condensation reaction has a yield of 99%, the overall synthetic yield approaches 60%. However, condensation reaction yields over 99% can not always be achieved because the condensation reaction depends on the sequence of the target peptides. As a result, amino acid deleted peptides accumulate as impurities derived from incomplete condensation reactions.
A capping by acetic anhydride is performed after every condensation reaction to terminate further elongation of peptide chains of non-target sequence and to avoid further production of amino acid deleted peptides. This procedure produces the effect that only the peptide having the target amino acid sequence has a free amino group at its N-terminus after the coupling of the final amino acid.
Several reports on the purification methods with use of the N-terminus amino group have been published (See, for example, T. J. Lobl, R. M. Deibel, and A. W. Yen, Anal. Biochem., 170, P.502 (1988)).
Another method has been developed in which the target peptide alone is absorbed and separated with a phenyl mercury column by attaching cysteine-methionine to the N-terminus of the synthesized target peptide, and using the SH group of the bound cysteine. Subsequent to the separation, the methionine-peptide bond is cleaved by BrCN to yield the target peptide (D. E. Krieger. B. W. Erikson, and R. B. Merrifield, Proc. Natl. Acad. Sci. U.S.A., 73, P.3160 (1976)). This method has a limitation of not being applicable to peptides containing methionine.
However, none of aforesaid methods has been able to achieve effective one-step separation. Instead they have required complicated processes.
The method of Lobl et al. selectively cleaves the t-BOC group as a protection group for the amino terminus of the target peptide synthesized on the solid support by using TFA/DCM, which is followed by washing with water, neutralizing, and washing with DCM and with DMF. The resulting peptide in a state of being protected with another functional group is then suspended in DMF. To the suspension, NHS-biotin (N-hydroxy succinimide biotin) is added in order to biotinylate the N-amino terminus of peptide. By using "low/high" HF, the biotinylated peptide is separated from the solid-phase support. The resultant mixture containing the biotinylated peptide is powdered by using ether, extracted with 6M guanidine-HCl buffer, and twice concentrated by dialysing. The resultant concentrate is passed through an avidin immobilized solid support such as avidin-agarose column (Pierce Co., Ltd.) in order to capture selectively the biotin-modified target peptide thereon. The biotin-modified target peptide can be eluted from the avidin immobilized solid support by using 0.1M guanidine-HCl (pH2.0). Thus, the method of Lobl et al. is no more than a separation method for a biotin-modified peptide or protein. It can never isolate the target peptide in its inherent or unmodified form, since there is no way for cleaving the biotin-peptide linkage selectively.
Improved methods are also known where NHS-SS-biotin (sulfosuccinimidyl 2-(biotin amide)ethyl-1,3-dithiopropionate) is used in place of NHS-biotin. After selective collection of the biotinylated peptide with an avidin-immobilized solid support, the SS bond of the reagent is broken by thiol so as to obtain the target peptide. However, the resulting peptide still contains an SH group residue from the reagent (See for example, Mouton C. A., et al. (1982) Arch. Biochem. Biophys. 218, 101-108; Shimkus, M., Levy, T. and Herman, T. (1985) Proc. Natl. Acad. Sci. (USA) 82, 2593-2597; Gretch, D. R, Suter, M. and Stinski, M. F. (1987) Anal. biochem. 163, PP.270-277).